The invention relates to plasmids, either in circular or linear form, useful in monitoring the efficiency of a restriction endonuclease digestion reaction.
Restriction endonucleases are a class of enzymes that occur naturally in bacteria and when they are purified away from other contaminating bacterial components, they can be used in the laboratory to break DNA molecules into precise fragments. They are the biochemical scissors by which genetic engineering and analysis is performed and, therefore, have proved to be indispensable tools in modern genetic research.
Restriction endonucleases act by recognizing and binding to particular sequences of nucleotides (the xe2x80x9crecognition sequencexe2x80x9d) along the DNA molecule. Once bound, they cleave the molecule within, or to one side of, the sequence. Different restriction endonucleases have affinity for different recognition sequences. Close to one hundred different restriction endonucleases have been identified among the many hundreds of baterial species that have been examined to date.
Plasmid construction is an essential technique in the cloning and manipulation of genes. Most plasmid construction strategies are designed to ensure that insertion of a DNA fragment into a vector predominates over the default event in which the vector recircularizes without incorporating an insert. There are four major ways of achieving high insertion ratios. They all rely, in one way or another, on rendering the vector incapable of circularizing without ligating to the insert fragment. For example, use of phosphatased vectors favors a high insertion frequency because dephosphorylated ends can only ligate to phosphorylated ends supplied by the insert. In utilizing this method, however, the phosphatase is difficult to control. TA vectors, used for cloning PCR-amplified DNA, are designed to be linearized in such a way as to leave single 3xe2x80x2 thymidine overhangs which can ligate only to adenine over-hangs like those left on the 3xe2x80x2 ends of DNA extended by taq (in this method ligation is catalyzed either by DNA ligase or DNA topoisomerase). The TA cloning polymerase method also favors efficient insertion of the PCR product. One limitation of this method is that not all thermostable DNA polymerases used for PCR leave the 3xe2x80x2 adenine. Partial fill-in of most 5xe2x80x2 restriction site overhangs by Klenow polymerase renders them non-circularizable; an insertable fragment is then generated by partial fill-in of an appropriate restriction site (e.g. a vector cleaved with SalI (G!TCGAC), filled in with T and C can be ligated to Sau3Al (!GATC) fragments filled in with G and A).
The fourth widely used technique is referred to as xe2x80x9cforced ligation.xe2x80x9d This technique makes use of a vector digested with two different restriction enzymes to generate a non-circularizable linear molecule; an insert fragment generated with the same enzymes is ligated in, thereby closing the plasmid. Forced ligation of gel-purifed restriction fragments is the simplest and most efficient method of plasmid construction in situations where segments are transferred from one plasmid to another, or connected together in new combinations, e.g. in mutagenesis, in analysis of gene expression, and in numerous other applications. Double-digested vectors are also convenient for cloning PCR fragments into a construct directly, without the necessity of a TA cloning step, provided that the PCR primers are designed with appropriate restriction sites.
The forced ligation method provides a high percentage of insertion (often greater than 90%), provided that both of the vector sites are fully digested, a criterion which is not always easy to achieve, since many enzymes are of uncertain stability or are inhibited by contaminants present in DNA preparations. Hence demonstration of good digestion is highly desirable before proceeding with the construction; this is also problematic, since complete digestion is impossible to document by agarose electrophoresis when the two sites are in a polylinker; and even when the sites are separated by as much as 10% of the full length of the vector, the desired double cut linear may not be fully resolved from contaminating partially digested molecules. Even a barely detectable residue of partially digested vector leads to an inordinately large number of re-circularized plasmids, requiring a large number of minipreps to be analyzed in search of the desired construct. This is especially troublesome when available insert fragments are present in limited amounts or when lower frequency events, such as triple ligations, are desired.
Because of the uncertainties in obtaining reliable double digests, marker plasmids can be included in mixed test digestions to be sure that both enzymes are active and that neither is inhibited by contaminants in the vector plasmid prep or in a PCR product to be inserted. Suitable marker plasmids can sometimes be found in a plasmid collection for a given pair of enzymes but often none is available, limiting the value of the digest marker maneuver, especially in labs with small plasmid inventories. What is needed is a double digest marker plasmid or set of plasmids which is informative of digestion efficiency for a variety of known restriction endonucleases.
In one aspect, the invention relates to a plasmid for use in monitoring the efficiency of a restriction endonuclease digestion, comprising at least one spacer segment comprising a nucleic acid sequence that is restriction site-free and at least two polylinker regions containing a plurality of unique restriction sites. The restriction sites are distributed so that digestion of the plasmid with two restriction endonucleases whose sites are represented on the plasmid results in two fragments, each of the fragments being sufficiently different in size from the intact plasmid so as to be readily distinguishable from the plasmid. Generally, fragments that are at least about 15% shorter than the intact plasmid can easily be distinguished from the intact plasmid when visualized on an agarose gel.
In another aspect, the invention relates to a plasmid for monitoring the digestion efficiency of a restriction endonuclease digestion comprising at least one spacer segment comprising a nucleic acid sequence that is restriction site-free and at least two polylinker regions where the polylinker regions contain a plurality of unique restriction sites distributed so that, for any two sites, the two sites are situated within different polylinker regions on at least one plasmid of a set of such plasmids. The polylinker regions are separated by a spacer region of restriction site-free nucleic acid whose length is about 15-85% of the length of the plasmid. Alternatively, the length of the spacer region of restriction site-free nucleic acid may be 20-85%, 30-85%, 40-85% or 50-85% of the length of the intact plasmid. Digestion of the plasmid with two endonucleases whose recognition sites are represented on the plasmid, therefore, results in two fragments, one of the fragments being at least about 15%-85% of the length of the intact plasmid. The plasmid of the present invention further comprises a replication origin and a selectable marker, for example, the ampR gene. In one embodiment, the plasmid of the present invention comprises a vector backbone of a plasmid such as pUC, pBR322 or pBS.
In a related aspect, the invention relates to a set of plasmids for use in monitoring the efficiency of a restriction endonuclease digestion, wherein each of the plasmids of the set comprises at least one spacer segment comprising a nucleic acid sequence that is restriction site-free and at least two polylinker regions containing a plurality of unique restriction sites distributed so that, for any two sites, the two sites are situated within different polylinker regions on at least one plasmid of the set and the polylinker regions are separated by a spacer segment whose length is about 15-85% of the length of the plasmid.
In yet another aspect, the invention relates to a method for designing a set of plasmids for use in monitoring the efficiency of a restriction endonuclease digestion comprising: (a) identifying at least one spacer segment comprising a nucleic acid sequence that is restriction site-free; (b) identifying a plurality of restriction sites to be represented on the plasmids; (c) assigning each restriction site to a polylinker region on one of the plasmids such that for any two restriction sites there is at least one polylinker in the set that does not contain both sites; (d) distributing the polylinker regions on the plasmids such that the polylinker regions are separated by a spacer segment at least about 15%-85% of the length of the plasmid.
In still another aspect, the invention relates to a method for designing a set of plasmids for monitoring the efficiency of a restriction endonuclease digestion comprising: (a) identifying at least one spacer segment comprising a nucleic acid sequence that is restriction site-free; (b) identifying a plurality of restriction sites to be represented on the plasmids; (c) determining the number of plasmids (b) and the number of polylinker regions (a) on each plasmid necessary to accommodate the desired restriction sites, wherein the maximum number (N) of sites which can be represented is N=ab, where a is the number of polylinkers in each plasmid and b is the number of plasmids in the set; (e) assigning each of the restriction sites to a polylinker region in accordance with a template, wherein the template corresponds to an axc3x97b matrix, and where each site is in a different polylinker from any other site in at least one of the plasmids in the set.
In one aspect, the invention relates to a method of designing a set of three plasmids for monitoring the efficiency of a restriction endonuclease digestion comprising (a) identifying at least two spacer segments comprising a nucleic acid sequence that is restriction site-free; (b) identifying 27 restriction sites to be represented on the three plasmids; (c) numerically ordering the 27 restriction sites; and (d) assigning each of the restriction sites to a polylinker region as follows:
(i) sites 1-9 are assigned to a first polylinker on a first plasmid, sites 10-18 are assigned to a second polylinker on the first plasmid and sites 19-27 are assigned to a third polylinker on the first plasmid;
(ii) sites 1, 4, 7, 10, 13, 16, 19, 22 and 25 are assigned to a first polylinker on a second plasmid, sites 2, 5, 8, 11, 14, 17, 20, 23, and 26 are assigned to a second polylinker on the second plasmid and sites 3, 6, 9, 12, 15, 18, 21, 24, and 27 are assigned to a third polylinker on the second plasmid;
(iii) sites 1, 2, 3, 10, 11, 12, 19, 20 and 21 are assigned to a first polylinker on a third plasmid, sites 4, 5, 6, 13, 14, 15, 22, 23 and 24 are assigned to a second polylinker on the third plasmid and sites 7, 8, 9, 16, 17, 18, 25, 26 and 27 are assigned to a third polylinker on the third plasmid.
The polylinker regions are then distributed on each of the plasmids such that the polylinker regions are separated from each other by a spacer segment at least about 15% of the length of the intact plasmid.
In a related aspect, the invention relates to a set of plasmids designed and constructed according to this method. The plasmids so constructed may further comprise additional restriction sites situated in non-polylinker regions of the plasmids.
In yet another aspect, the invention relates to a method of constructing a set of three plasmids for monitoring the efficiency of a restriction endonuclease digestion comprising:
(a) identifying at least one spacer segment comprising a nucleic acid sequence that is restriction site-free; (b) identifying 64 restriction sites to be represented on the three plasmids; (c) numerically ordering said restriction sites; and (d) assigning each of said restriction sites to a polylinker region as follows:
(i) sites 1-16 are assigned to a first polylinker on a first plasmid, sites 17-32 are assigned to a second polylinker on the first plasmid, sites 33-48 are assigned to a third polylinker on the first plasmid and sites 49-64 are assigned to a fourth polylinker on the first plasmid;
(ii) sites 1-4, 29-32, 41-44 and 53-56 are assigned to a first polylinker on a second plasmid, sites 5-8, 17-20, 45-48 and 57-60 are assigned to a second polylinker on the second plasmid, sites 9-12,21-24, 33-36 and 61-64 are assigned to a third polylinker on the second plasmid and sites 13-16, 25-28, 37-40 and 49-52 are assigned to a fourth polylinker on the second plasmid;
(iii) every fourth site beginning with site number 1 is assigned to a first polylinker on a third plasmid, every fourth site beginning with site number 2 is assigned to a second polylinker on the third plasmid, every fourth site beginning with site number 3 is assigned to a third polylinker on the third plasmid; and every fourth site beginning with site number 4 is assigned to a fourth polylinker on the third plasmid. The polylinker regions are then distributed on each of the plasmids such that they are separated by a spacer segment at least about 15% of the length of the plasmid.
In a related aspect, the invention relates to a set of three plasmids constructed according to this method and includes plasmids in which additional restriction sites have been placed in non-polylinker regions of the plasmid.
In yet another aspect, the invention relates to a method for preparing plasmids for monitoring the digestion efficiency of a restriction endonuclease digest reaction, comprising the steps of: (a) transfecting host cells with the monitor plasmid described above; (b) growing the cells under conditions to provide a quantity of cells containing the plasmids; and (c) purifying the plasmids from the cells.
In still another aspect, the invention relates to a method for monitoring the digestion efficiency of a restriction endonuclease digestion reaction comprising: (a) adding a monitor plasmid of the type described above to a sample preparation of a non-monitor plasmid; (b) initiating an endonuclease digestion reaction; (c) allowing the reaction to proceed to completion; and (d) determining, usually by gel electrophoresis of the digested material, whether the monitor plasmid has been digested, wherein digestion of the monitor plasmid indicates digestion of non-monitor plasmids in the sample preparation.
In yet another aspect, the invention relates to a kit for monitoring the digestion efficiency of a restriction endonuclease digestion reaction comprising at least one plasmid where the plasmid contains a plurality of unique restriction sites distributed so that digestion of the plasmid with any two restriction endonucleases represented on the plasmid results in two plasmid fragments, one of the fragments being at least about 15% of the length of the undigested plasmid. The kit may comprise one, two, three, four or more plasmids, as required and includes instructions for use of plasmid(s). The kit may additional include restriction endonucleases whose sites are represented on the plasmid(s) and appropriate buffers.